Method of measuring lipoarabinomannan and application thereof

ABSTRACT

A method for measuring LAM and a method for detecting an acid-fast bacterium, which comprise at least a step of allowing a  Limulus  reagent to contact with a LAM-containing sample; a method for removing reactivity of LAM with a  Limulus  reagent, which comprises at least a step of allowing a predetermined substance to coexist with a LAM-containing sample; a method for measuring Et in a LAM-containing sample and a method for detecting an Et-related disease, which each is a method for measuring an endotoxin using a  Limulus  reagent, which comprises at least a step of removing reactivity of LAM with a  Limulus  reagent by the above-described removing method; a method for measuring BG in a LAM-containing sample and a method for detecting mycosis, which is a method for measuring BG using a  Limulus  reagent, which comprises at least a step of removing reactivity of LAM with a  Limulus  reagent by the described-described removing method; and the like.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of application Ser. No. 10/584,071 filed Jun. 22,2006 (now U.S. Pat. No. 7,851,179 B2 issued Dec. 14, 2010), which is aNational Stage of PCT/JP2004/019206 filed Dec. 22, 2004 which claimspriority from Japanese Application No. 2003-425472 filed Dec. 22, 2003,the disclosures of which are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to a method for measuringlipoarabinomannan, a kit to be used therein, a method for removingreactivity of lipoarabinomannan with a Limulus reagent, a method formeasuring endotoxin and (1→3)-β-glucan using the same, a kit for usetherein, an agent for binding of lipoarabinomannan and the like.

BACKGROUND ART

A Limulus reagent (also called a lysate reagent) is a reagent whichcomprises a horseshoe crab amoebocyte lysate as the main component andis used for the detection and measurement of an endotoxin (hereinafterreferred to as “Et”) and a (1→3)-β-glucan (hereinafter referred to as“BG”). Since Et and BG have reactivity with a Limulus reagent, when theLimulus reagent and there substances contact with one another, a cascadereaction in which various factors in the Limulus reagent are concerned(hereinafter referred to as “Limulus reaction”) is induced, so thatthese substances can be detected and measured by detecting thisreaction.

On the other hand, it is known that a lipoarabinomannan (hereinafterreferred to as “LAM”) is a cell wall component specific to acid-fastbacteria (e.g., tubercle bacillus, etc.).

Patent Reference 1 discloses a Limulus reaction-activating substancewhich is physically different from Et and BG, an inactivation methodthereof, a measuring method thereof and the like. However, thissubstance is completely different from LAM as is described later, andthere is no disclosure or suggestion that LAM has reactivity with aLimulus reagent.

-   Patent Reference 1: JP-A-10-185924

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method for measuringLAM, a kit to be used therein, a method for removing reactivity of LAMwith Limulus reagent, a method for measuring Et and BG using the same, akit for use therein, an agent for binding of LAM and the like.

Means for Solving the Problems

The present inventors have conducted intensive studies in order to solvethe above-described problems and found as a result for the first timethat LAM has reactivity with a Limulus reagent and, based on thisfinding, have provided a method for measuring LAM, a kit to be usedtherein, a method for removing reactivity of LAM with Limulus reagent, amethod for measuring Et and BG using the same, a kit for use therein andthe like.

That is, the present invention provides a method for measuring LAM in asample, which comprises at least a step of allowing a Limulus reagent tocontact with a “LAM-containing sample” (hereinafter referred to as“LAM-measuring method of the present invention”). It is preferable thatthis method further comprises a step of heating the “LAM-containingsample” before the contact with the Limulus reagent. In addition, it ispreferable that the Limulus reagent is an Et-specific Limulus reagent.

Also, the present invention provides a method for detecting an acid-fastbacterium, which comprises using the LAM-measuring method of the presentinvention (hereinafter referred to as “acid-fast bacterium-detectingmethod of the present invention”). It is preferable that the acid-fastbacterium to be detected is a tubercle bacillus.

Also, the present invention provides a kit for measuring LAM, whichcomprises a Limulus reagent as a component (hereinafter referred to as“LAM-measuring kit of the present invention”). It is preferable that theLimulus reagent is an Et-specific Limulus reagent.

Also, the present invention provides a kit for detecting an acid-fastbacterium, which comprises the LAM-measuring kit of the presentinvention (hereinafter referred to as “acid-fast bacterium detection kitof the present invention”). It is preferable that the acid-fastbacterium to be detected is a tubercle bacillus.

Also, the present invention provides a method for removing reactivity ofLAM in a “LAM-containing sample” with a Limulus reagent, which comprisesat least a step of allowing one or more substance(s) selected from thefollowing group to coexist with the sample (hereinafter referred to as“reactivity-removing method of the present invention”):

a surfactant, an anti-tuberculosis antibody, an anti-LAM antibody, BG, acarboxymethylated BG, a factor G activation inhibitor, a strong alkalinesubstance, polymyxin B, colistin, concanavalin A, histidine andhistamine.

Also, the present invention provides a method for measuring Et in a“LAM-containing sample”, which is a method for measuring Et using aLimulus reagent, which comprises at least a step of removing reactivityof LAM with a Limulus reagent by the reactivity-removing method of thepresent invention (hereinafter referred to as “Et-measuring method ofthe present invention”). It is preferable that the Limulus reagent is anEt-specific Limulus reagent.

Also, the present invention provides a method for detecting anEt-related disease, which comprises using the Et-measuring method of thepresent invention (hereinafter referred to as “Et-related diseasedetection method of the present invention”).

Also, the present invention provides a kit for measuring Et, whichcomprises a Limulus reagent and one or more substance(s) selected fromthe following group as components (hereinafter referred to as“Et-measuring kit of the present invention”):

a surfactant, an anti-tuberculosis antibody, an anti-LAM antibody, BG, acarboxymethylated BG, a factor G activation inhibitor and a strongalkaline substance.

It is preferable that the Limulus reagent is an Et-specific Limulusreagent.

Also, the present invention provides a kit for detecting an Et-relateddisease, which comprises the Et-measuring kit of the present invention(hereinafter referred to as “Et-related disease detection of the presentinvention”).

Also, the present invention provides a method for measuring BG using aLimulus reagent in a “LAM-containing sample”, which comprises at least astep of removing reactivity of LAM with a Limulus reagent by thereactivity-removing method of the present invention (hereinafterreferred to as “BG-measuring method of the present invention”). It ispreferable that the Limulus reagent is a BG-specific Limulus reagent.

Also, the present invention provides a method for detecting mycosis,which comprises using the BG-measuring method of the present invention(hereinafter referred to as “mycosis detection method of the presentinvention”).

Also, the present invention provides a kit for measuring BG, whichcomprises a Limulus reagent and one or more substance(s) selected fromthe following group as components (hereinafter referred to as“BG-measuring kit of the present invention”):

a surfactant, an anti-tuberculosis antibody, an anti-LAM antibody, astrong alkaline substance, polymyxin B, colistin, concanavalin A,histidine and histamine.

Also, the present invention provides a kit for detecting mycosis, whichcomprises the BG-measuring kit of the present invention (hereinafterreferred to as “mycosis detection kit of the present invention”).

Also, the present invention provides an agent for binding of LAM, whichcomprises one or more substance(s) selected from the following group asan active ingredient (hereinafter referred to as “binder of the presentinvention”):

an anti-tuberculosis antibody, an anti-LAM antibody, (1→3)-β-glucan, acarboxymethylated (1→3)-β-glucan, a factor G activation inhibitor,polymyxin B, colistin, concanavalin A, histidine and histamine.

Effect of the Invention

The LAM-measuring method of the present invention, the acid-fastbacterium detection method of the present invention, the LAM-measuringkit of the present invention and the acid-fast bacterium detection kitof the present invention can be used for measuring and detecting LAM andacid-fast bacteria conveniently, quickly and inexpensively and thereforeare markedly useful.

In addition, the reactivity-removing method of the present invention canbe used for removing influence of LAM in the Limulus reactionconveniently, quickly and inexpensively and therefore is markedlyuseful.

The Et-measuring method of the present invention, the Et-related diseasedetection method of the present invention, the Et-measuring kit of thepresent invention and the Et-related disease detection kit of thepresent invention can be used for measuring and detecting Et andEt-related diseases specifically and also conveniently, quickly andinexpensively and therefore are markedly useful.

The BG-measuring method of the present invention, the mycosis detectionmethod of the present invention, the BG-measuring kit of the presentinvention and the mycosis detection kit of the present invention can beused for measuring and detecting BG and mycosis specifically and alsoconveniently, quickly and inexpensively and therefore are markedlyuseful.

The agent for binding of the present invention can be used for detectingand measuring LAM and for removing LAM and therefore is markedly useful.

BEST MODE FOR CARRYING OUT THE INVENTION

<1> LAM-Measuring Method of the Present Invention

The LAM-measuring method of the present invention is a method formeasuring LAM in a sample, which comprises at least a step of allowing aLimulus reagent to contact with “the LAM-containing sample”.

The “Limulus reagent” is not particularly limited, so long as it is areagent which comprises a horseshoe crab amoebocyte lysate as the maincomponent. The kind of this horseshoe crab is also not limited, and theamoebocyte lysate of any one of Limulus polyphemus (North Americanhorseshoe crab) and Tachypleus tridentatus, Tachypleus gigas andTachypleus rotundicauda (Asian horseshoe crab) can be used. Theamoebocyte lysate can be produced by a conventionally known method. Inaddition, a Limulus reagent which is on the market may be used.

This Limulus reagent may be a Limulus reagent prepared in such a mannerthat it does not react with BG (referred to as “Et-specific Limulusreagent” in this specification) or a Limulus reagent which reacts withEt and BG, but it is preferably an “Et-specific Limulus reagent”. ThisEt-specific Limulus reagent can be produced by a conventionally knownmethod, or a commercially available product may be used.

The “LAM-containing sample” is not particularly limited, so long as itis a sample that contains LAM or has a possibility of containing LAM.Since LAM is a cell wall component specific to acid-fast bacteria,living cells or dead cells themselves of an acid-fast bacterium (e.g.,tubercle bacillus, etc.), cell walls thereof, a sample containing thecell wall component or having a possibility of containing the same canbe exemplified. Examples of such a sample include tubercle bacillusvaccine and the like.

In addition, a “sample derived from the living body” can also be used asthe “LAM-containing sample”. The “sample derived from the living body”is not particularly limited too, but a body fluid is preferable. Thebody fluid is not particularly limited, so long as it is a body fluidthat contains LAM or has a possibility of containing the same. Examplesinclude blood (in this specification, this is used as a general ideaincluding serum and plasma), urine, sweat, saliva, tears, synovialfluid, expectoration, milk, spinal fluid and the like. Among these,blood is preferable.

In this connection, when blood is used as the “sample derived from theliving body”, it is preferable to remove or inactivate Limulusreaction-interfering factors in blood (serine protease, serine proteaseinhibitor, etc.) in advance by a conventionally known method (e.g., themethod described in JP-A-58-85162, etc.).

In addition, the method for “contacting” a Limulus reagent with a sampleis also not limited, so long as a factor in the Limulus reagent isallowed to contact with a LAM molecule in the sample, so that the samplemay be added to the Limulus reagent, the Limulus reagent may be added tothe sample, or both may be added at the same time.

The LAM-measuring method of the present invention may contain anotherstep, so long as it contains the step of allowing a Limulus reagent anda “LAM-containing sample” to contact with each other. For example, it ispreferable that it further comprises a step of heating the“LAM-containing sample” before its contact with the Limulus reagent.According to the specification, the “heating” means that a substanceunder room temperature is heated. The temperature after heating is notparticularly limited, but the heating is preferably carried out at from37 to 121° C., preferably from 60 to 100° C., particularly 95° C. Theperiod of time for keeping the heated state is not particularly limitedtoo, but it is preferable to keep the heated state for from 5 to 60minutes, preferably from 10 to 30 minutes, and particularly about 20minutes.

When a Limulus reagent is contacted with a sample, LAM in the sampleactivates the factor C (known as a factor which is activated by Et) inthe Limulus reagent, and a Limulus reaction is induced thereby.

By detecting and measuring this Limulus reaction, LAM in the sample canbe measured. The Limulus reaction can be detected and measured byconventionally known methods. For example, chromogenic assay (endpointassay or kinetic assay), gel-clot assay, turbidimetric assay (endpointassay or kinetic assay) and the like conventionally known methods can beemployed as the detecting and measuring methods corresponding torespective methods.

In this connection, the “measurement” as used herein is a general ideawhich includes not only quantitative measurement but also qualitativemeasurement (measurement in the presence or absence of LAM, etc.).

As the quantitative measurement of LAM, various methods can be employedin response to the object. For example, strict determination can becarried out by preparing a calibration curve or a relational expressionon the relationship between the LAM concentration and the strength ofLimulus reaction using a sample having already known LAM concentrationand using this. In addition, when strict determination is not necessary,amounts of LAM between samples may be compared using two or moresamples. Since LAM induces a Limulus reaction, the amount of LAM in asample is large when strength of the Limulus reaction is high.

LAM can be qualitatively measured by detecting the presence or absenceof the Limulus reaction. Since LAM induces a Limulus reaction, LAM ispresent in a sample when the Limulus reaction is detected.

<2> Acid-Fast Bacteria Detection Method of the Present Invention

The acid-fast bacteria detection method of the present invention is amethod for detecting an acid-fast bacterium, which comprises using theLAM-measuring method of the present invention.

The acid-fast bacteria detection method of the present invention is amethod in which the LAM-measuring method of the present invention isdirectly applied to the detection of acid-fast bacteria. Since LAM is acell wall component specific to acid-fast bacteria, detection of anacid-fast bacterium can be carried out by measuring LAM.

The LAM-measuring method of the present invention should be referred toas the above-described <1>. According to the acid-fast bacteriadetection method of the present invention, a sample containing anacid-fast bacterium or having a possibility of containing an acid-fastbacterium is used as the “LAM-containing sample” of the LAM-measuringmethod of the present invention.

The bacteria to be detected are not particularly limited, so long asthey are bacteria classified as acid-fast bacteria. Examples includebacteria belonging to the genus Mycobacterium, the genus Nocardia, thegenus Rhodococcus, the genus Gordonia, the genus Corynebacterium and thelike. Among these, a tubercle bacterium (belonging to the genusMycobacterium) is preferable.

In addition, the acid-fast bacterium to be detected may be a living cellor dead cell.

Acid-fast bacteria in such a sample can be detected by using theLAM-measuring method of the present invention.

In this connection, the “detection” of acid-fast bacteria as used hereinis a general idea which includes not only a qualitative detection(detection of the presence or absence of acid-fast bacteria) but also aquantitative detection (detection of the amount of acid-fast bacteria,detection of the malignancy of acid-fast bacteria infection, etc.).

The acid-fast bacteria can be qualitatively measured by detecting thepresence or absence of a Limulus reaction. Since LAM as a cell wallcomponent specific to acid-fast bacteria induces a Limulus reaction, anacid-fast bacterium is present in the sample when the Limulus reactionis detected.

As the quantitative measurement of an acid-fast bacterium, variousmethods can be employed in response to the object. For example, strictdetermination can be carried out by preparing a calibration curve or arelational expression on the relationship between the amount of theacid-fast bacterium and the strength of Limulus reaction using a samplehaving already known acid-fast bacterium amount and using this. Inaddition, when strict determination is not necessary, amount of theacid-fast bacterium between samples may be compared using two or moresamples. Since LAM which is a cell wall component specific for acid-fastbacteria induces a Limulus reaction, amount of the acid-fast bacteriumin a sample is large when strength of the Limulus reaction is high.

<3> LAM-Measuring Kit of the Present Invention

The LAM-measuring kit of the present invention is a kit for measuringLAM, which comprises a Limulus reagent as a component. Explanation for“Limulus reagent” is the same as in the above-described <1>. That is, itis preferable that this Limulus reagent is also an Et-specific Limulusreagent.

The LAM-measuring kit of the present invention may further containanother component, so long as it comprises at least a “Limulus reagent”as a component. For example, distilled water for blank test use, areaction reagent solution, a buffer for reaction use and the like can becited as such a component. In addition, the LAM-measuring kit of thepresent invention can also contain a positive control (QC control) orthe like for the purpose of keeping the practice level between measuringbatches at a certain level.

These components can be preserved by containing in respectively separatecontainers.

LAM using the LAM-measuring kit of the present invention can be measuredin accordance with the LAM-measuring method of the present inventiondescribed in the above-described <1>.

<4> Acid-Fast Bacteria Detection Kit of the Present Invention

The acid-fast bacteria detection kit of the present invention is a kitfor detecting an acid-fast bacterium, which comprises the LAM-measuringkit of the present invention. Explanation for the LAM-measuring kit ofthe present invention should be referred to the above-described <3>.

Explanation for the acid-fast bacteria to be detected by acid-fastbacteria detection kit of the present invention is the same as in theabove-described acid-fast bacteria detection method of the presentinvention of the above-described <2>. That is, it is preferable that theacid-fast bacterium to be detected is a tubercle bacillus.

Acid-fast bacteria using the acid-fast bacteria detection kit of thepresent invention can be detected in accordance with the acid-fastbacteria detection method of the present invention of theabove-described <2>.

<5> Reactivity Removal Method of the Present Invention

The reactivity-removing method of the present invention is a method forremoving reactivity of LAM in a “LAM-containing sample” with a Limulusreagent, comprising at least a step of allowing one or more substance(s)selected from the following group to coexist with the sample:

a surfactant, an anti-tuberculosis antibody, an anti-LAM antibody, BG, acarboxymethylated BG, a factor G activation inhibitor, a strong alkalinesubstance, polymyxin B, colistin, concanavalin A, histidine andhistamine.

Explanation for the “LAM-containing sample” and “Limulus reagent” arethe same as in the LAM-measuring method of the present invention of theabove-described <1>.

The “surfactant” which can be used herein is not particularly limited,so long as it does not spoil the reactivity of Et and BG with theLimulus reagent, and it does not exert inhibitory action upon variousfactors which are present in the Limulus reagent and concerned in theLimulus reaction. For example, it may be any one of a cationicsurfactant, an anionic surfactant, an amphoteric surfactant, a nonionicsurfactant, a natural surfactant and the like.

Among these, it is preferable to select a nonionic surfactant which hasless direct action upon Et. In this connection, these surfactants may beused by optionally combining them.

Among nonionic surfactants, a surfactant having a structure in which itshydrophilic moiety has a polyoxyethylene (hereinafter also referred toas “polyoxyethylenes”) is preferable. As the polyoxyethylenes, apolyoxyethylene alkyl ether (represented by a formulaC_(n)N_(2n+1)(OCH₂CH₂)_(x)OH, and generally abbreviated and described asC_(n)E_(x)), a polyoxyethylene alkyl phenyl ether in which a phenylgroup is inserted between an alkyl chain and a polyoxyethylene chain(C_(n)ΦE_(x)) and an acyl polyoxyethylene sorbitan (C_(n)sorbitanE_(x))and the like can be cited, and these are called by general names (tradenames) of Brij (C_(n)E_(x)), Tergitol (C_(n)E_(x)), Triton X(C_(n)ΦE_(x)) and Tween (C_(n)sorbitanE_(x)), respectively, andgenerally used for many purposes such as solubilization of membraneproteins.

The polyoxyethylene chain (the “(OCH₂CH₂)_(x)OH” moiety in theabove-described formula, also referred to as “Ex”) of thepolyoxyethylenes which can be used herein is not particularly limited,but a compound has preferably an integer of x=2 to 25, more preferablyan integer of x=4 to 23, and most preferably an integer of x=7 to 13. Inaddition, the number of carbon atoms of the alkyl group (the“C_(n)H_(2n+1)” moiety described above, also referred to as “Cn”) of thepolyoxyethylenes which are used herein is not particularly limited, buta compound having an integer of n=8 to 18 is preferable.

Examples of such polyoxyethylenes include polyoxyethylene dodecyl ether,polyoxyethylene hexadecyl ether (also called polyoxyethylene cetylether), polyoxyethylene isooctyl phenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitolester and the like. Among these, polyoxyethylene hexadecyl ether ismarkedly preferable. In addition, it is preferable that thesesurfactants are used as aqueous solutions and have a certain micellesize.

In this connection, the solvent of these aqueous surfactant solutionsmay be a buffer. The buffer is preferably a buffer adjusted to a pHvalue of approximately from 7 to 9, and examples include Good's buffer[e.g., HEPES (N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acidbuffer), cholamine chloride buffer, BES buffer, MOPS buffer, TES buffer,HEPPS buffer (N-2-hydroxyethyl-piperazine-N′-3-propanesulfonic acid),Tricine buffer, glycinamide buffer, Bicine buffer, TAPS buffer or thelike], Tris-HCl buffer and the like.

An amount of the surfactant to be coexisted with the “LAM-containingsample” can be optionally changed according to the kind and the like ofthe surfactant and therefore is not particularly limited. As specificconcentration of the surfactant, generally from 0.001% to 0.8% (w/v),preferably from 0.003% to 0.5% (w/v), more preferably from 0.005% to0.3% (w/v) and the like, as the final concentration when contacted withthe “LAM-containing sample”, can be exemplified.

Also, the “anti-tuberculosis antibody” which can be used herein is notparticularly limited, so long as it is an antibody which binds to theLAM existing in the cell wall of a tubercle bacillus, and it may beproduced by a conventionally known method using a tubercle bacillus, acell wall component thereof or the like as the antigen, or may becommercially available. Among these, an antibody which specificallybinds to the LAM existing in the cell wall of a tubercle bacillus ispreferable.

In addition, the “anti-LAM antibody” which can be used herein is notparticularly limited, so long as it is an antibody which binds to LAM,and it may be produced by a conventionally known method using LAM as theantigen, or may be commercially available. Among these, an antibodywhich specifically binds to LAM is preferable.

These antibodies may be not only those which completely maintain themolecular structure of immunoglobulin as a matter of course, but alsothose which are made into fragments containing Fab by treating with aprotease (e.g., plasmin, pepsin, papain, etc.) that does not degrade theantigen-binding site (Fab). Examples of the fragment containing Fab ofantibody include Fabc, (Fab′)₂ and the like, in addition to Fab.

In addition, when nucleotide sequences of the genes encoding theseantibodies or amino acid sequences of these antibodies are determined,Fab-containing fragments of these antibodies or chimeric antibodiesthereof can also be prepared by genetic engineering techniques. SuchFab-containing fragments of these antibodies or chimeric antibodiesthereof are also included in the general idea of “antibody” according tothis specification.

Amounts of these antibodies to be coexisted with the “LAM-containingsample” can be optionally changed according to the kind and the like ofeach antibody and therefore are not particularly limited.

Also, the “BG” which can be used herein is also not particularlylimited, and examples include but pachyman, curdlan, CSBG (BG derivedfrom Candida albicans cells) and the like. Among these, pachyman ispreferable. In addition, the BG may not only contain β-1,3 bond alonebut also have a branch through β-1,6 bond or the like.

In addition, the BG may be a derivative in which a functional group orthe like is modified. Examples of the derivative includecarboxymethylated BG. Particularly, carboxymethylcurdlan is preferable.

Amounts of the BG and a derivative thereof to be coexisted with the“LAM-containing sample” can be optionally changed in response to theirkinds, molecular sizes and the like and therefore are not particularlylimited.

In addition, the “factor G activation inhibitor” which can be usedherein is not particularly limited too, so long as it is a substancehaving the action to inhibit activation of the factor G which is presentin the Limulus reagent, and examples include the polyglucoside and thelike described in WO 90/02951. An amount of the “factor G activationinhibitor” to be coexisted with the “LAM-containing sample” can beoptionally changed according to the kind and the like of the “factor Gactivation inhibitor” and therefore is not particularly limited.

In addition, the “strong alkaline substance” which can be used herein isnot particularly limited too, but an alkali metal hydroxide ispreferable. The alkali metal hydroxide includes sodium hydroxide,potassium hydroxide and the like.

Also, as the “polymyxin B”, “colistin”, “concanavalin A”, “histidine”and “histamine” which can be used herein, those which are on the marketcan also be used.

The order, method and the like in allowing these substances to coexistwith the “LAM-containing sample” are not particularly limited, so longas these substances can be present in the “LAM-containing sample”without causing their modification or destruction.

The method for allowing these substances to coexist with the“LAM-containing sample” is attained in general by thoroughly mixing the“LAM-containing sample” with these substances. Also, regarding the orderof allowing these substances to coexist with the “LAM-containingsample”, these substances may be added to the Limulus reagent, and theirmixing and reaction with the “LAM-containing sample” may be carried outat the same time, but it is preferable from the viewpoint of the effectthat they are mixed with the “LAM-containing sample” in advance prior tothe contact of the Limulus reagent with the “LAM-containing sample”.

By the reactivity-removing method of the present invention, reactivityof the LAM in the “LAM-containing sample” with the Limulus reagent canbe specifically removed.

<6> Et-Measuring Method of the Present Invention

The Et-measuring method of the present invention is a method formeasuring Et using a Limulus reagent in a “LAM-containing sample”, whichcomprises at least a step of removing reactivity of LAM with a Limulusreagent by the reactivity-removing method of the present invention.

Explanations for the “Limulus reagent” and the “LAM-containing sample”are the same as in the above-described <1> LAM-measuring method of thepresent invention. That is, it is preferable that this Limulus reagentis also an Et-specific Limulus reagent.

Also, the reactivity-removing method of the present invention should bereferred to the above-described <5>. However, it is necessary to use asubstance other than polymyxin B, colistin, concanavalin A, histidineand histamine in this case.

The Et-measuring method of the present invention may contain othersteps, so long as it is a method for measuring Et using a Limulusreagent, wherein it comprises a step of removing reactivity of LAM witha Limulus reagent by the reactivity-removing method of the presentinvention.

In addition, the timing for removing reactivity of LAM with a Limulusreagent by the reactivity-removing method of the present invention isalso not limited, but it is preferable to arrange the step of removingreactivity of LAM with a Limulus reagent by the reactivity-removingmethod of the present invention before the contact of the Limulusreagent with the “LAM-containing sample”. Explanation for the “contact”as used herein is the same as in the above-described <1> LAM-measuringmethod of the present invention.

According to the Et-measuring method of the present invention, thereactivity of LAM in the “LAM-containing sample” with a Limulus reagentis removed, so that Et in the sample can be measured without influenceof LAM.

The Limulus reaction induced by Et can be detected or measured by aconventionally known method. For example, conventionally known methods,such as chromogenic assay (endpoint assay or kinetic assay), gel-clotassay and turbidimetric assay (endpoint assay or kinetic assay), can beemployed as the detecting and measuring methods corresponding torespective methods.

As described above, the “measurement” as used in this specification is ageneral idea which includes not only quantitative measurement but alsoqualitative measurement (measurement of the presence or absence of Et,etc.).

As the quantitative measurement of Et, various methods can be employedin response to the object. For example, strict determination can becarried out by preparing a calibration curve or a relational expressionon the relationship between the Et concentration and the strength ofLimulus reaction using a sample having already known Et concentrationand using this. In addition, when strict determination is not necessary,amounts of Et between samples may be compared using two or more samples.Since Et induces a Limulus reaction, the amount of Et in a sample islarge when strength of the Limulus reaction is high.

Et can be qualitatively measured by detecting the presence or absence ofthe Limulus reaction. Since Et induces a Limulus reaction, Et is presentin a sample when the Limulus reaction is detected.

<7> Et-Related Disease Detection Method of the Present Invention

The Et-related disease detection method of the present invention is amethod for detecting an Et-related disease, which comprises using theEt-measuring method of the present invention.

The Et-related disease detection method of the present invention is amethod in which the Et-measuring method of the present invention isdirectly applied to the detection of Et-related diseases.

The Et-measuring method of the present invention should be referred toas the above-described <6>. According to the Et-related diseasedetection method of the present invention, a living body-derived samplecontaining Et or having a possibility of containing Et is used as the“LAM-containing sample” of the Et-measuring method of the presentinvention.

The Et-related disease to be detected is not particularly limited, solong as it is a disease which is caused based on Et. Examples includeendotoxemia and Gram-negative bacterial infection.

In addition, the “sample derived from the living body” is notparticularly limited too, but a body fluid is preferable. The body fluidis not particularly limited, so long as it is a body fluid that containsEt or has a possibility of containing the same. Examples include blood(according to this specification, this is used as a general idea whichincludes serum and plasma too), urine, sweat, saliva, tears, synovialfluid, expectoration, milk, spinal fluid and the like. Among these,blood is preferable.

In this connection, when blood is used as the “sample derived from theliving body”, it is preferable to remove or inactivate Limulusreaction-interfering factors in blood (serine protease, serine proteaseinhibitor, etc.) in advance by a conventionally known method (e.g., themethod described in JP-A-58-85162, etc.)

An Et-related disease can be detected in such a sample by using theEt-measuring method of the present invention.

As described above, the “detection” as used in this specification is ageneral idea which includes not only qualitative detection (detection ofthe presence or absence of an Et-related disease) but also quantitativemeasurement (detection of the malignancy of an Et-related disease,etc.).

An Et-related disease can be qualitatively measured by detecting thepresence or absence of a Limulus reaction. Since Et induces the Limulusreaction, an Et-related disease is caused or there is a possibility ofthe Et-related disease when the Limulus reaction is detected.

As the quantitative measurement of an Et-related disease, variousmethods can be employed in response to the object. For example, strictdetermination can be carried out by preparing a calibration curve or arelational expression on the relationship between the Et concentrationand the strength of Limulus reaction using a sample having already knownEt concentration and using this. In addition, when strict determinationis not necessary, amounts of Et between samples may be compared usingtwo or more samples. Since Et induces a Limulus reaction, the amount ofEt in a sample is large when the strength of the Limulus reaction ishigh, so that this can be correlated with a fact that malignancy of theEt-related disease is high or there is a possibility thereof.

<8> Et-Measuring Kit of the Present Invention

The Et-measuring kit of the present invention is a kit for measuring Et,which comprises a Limulus reagent and one or more substance(s) selectedfrom the following group as components:

a surfactant, an anti-tuberculosis antibody, an anti-LAM antibody, BG, acarboxymethylated BG, a factor G activation inhibitor and a strongalkaline substance.

Explanations for the “Limulus reagent” and the above-described varioussubstances are the same as in the above-described <1> and <5>. That is,it is preferable that this Limulus reagent to be used herein is also anEt-specific Limulus reagent.

The Et-measuring kit of the present invention may further containanother component, so long as it contains at least a “Limulus reagent”as a component. Examples of such a component include distilled water forblank test use, a reaction reagent solution, a buffer for reaction useand the like. In addition, the Et-measuring kit of the present inventioncan also contain a positive control (QC control) or the like for thepurpose of keeping the practice level between measuring batches at acertain level.

These components can be preserved by containing in respectively separatecontainers.

Et using the Et-measuring kit of the present invention can be measuredin accordance with the Et-measuring method of the present inventiondescribed in the above-described <6>.

<9> Et-Related Disease Detection Kit of the Present Invention

The Et-related disease detection kit of the present invention is a kitfor detecting an Et-related disease, which comprises the Et-measuringkit of the present invention. Explanation for the Et-measuring kit ofthe present invention should be referred to the above-described <8>.

Explanation for the Et-related disease to be detected by the Et-relateddisease detection kit of the present invention and explanation of the“sample derived from the living body” and the like are the same as inthe Et-related disease detection method of the present invention in theabove-described <7>. That is, the Et-related disease to be detectedincludes endotoxemia and Gram-negative bacterial infection, and it ispreferable that the “sample derived from the living body” is blood. Inaddition, it is also the same as in the above-described <7> that whenblood is used as the “sample derived from the living body”, it ispreferable to remove or inactivate Limulus reaction-interfering factorsin blood (serine protease, serine protease inhibitor, etc.) in advance.

An Et-related disease using the Et-related disease detection kit of thepresent invention can be detected in accordance with the Et-relateddisease detection method of the present invention described in theabove-described <7>.

<10> BG-Measuring Method of the Present Invention

The BG-measuring method of the present invention is a method formeasuring BG using a Limulus reagent in a “LAM-containing sample”, whichcomprises at least a step of removing reactivity of LAM with a Limulusreagent by the reactivity-removing method of the present invention.

Explanations for the “Limulus reagent” and the “LAM-containing sample”are the same as in the LAM-measuring method of the present invention ofthe above-described <1>.

In addition, explanation for the reactivity-removing method of thepresent invention should be referred to the above-described <5>.However, it is necessary in this case to use a substance other than BG,a carboxymethylated BG and a factor G activation inhibitor.

The BG-measuring method of the present invention may contain othersteps, so long as it is a method for measuring BG using a Limulusreagent wherein it comprises a step of removing reactivity of LAM with aLimulus reagent by the reactivity-removing method of the presentinvention.

In addition, explanations of the timing for applying thereactivity-removing method of the present invention and of the “contact”and the like are the same as in the Et-measuring method of the presentinvention of the above-described <6>.

According to the BG-measuring method of the present invention, thereactivity of LAM in the “LAM-containing sample” with a Limulus reagentis removed, so that BG in the sample can be measured without influenceof LAM.

Explanation for the detection and measurement of the Limulus reactioninduced by BG, explanation of the “measurement” and the like are alsothe same as in the Et-measuring method of the present invention of theabove-described <6>.

<11> Mycosis Detection Method of the Present Invention

The mycosis detection method of the present invention is a method fordetecting mycosis, which comprises using the BG-measuring method of thepresent invention.

In the mycosis detection method of the present invention, theBG-measuring method of the present invention is directly applied to thedetection of mycosis.

Explanation for the BG-measuring method of the present invention shouldbe referred to the above-described <10>. In the mycosis detection methodof the present invention, a sample derived from the living body whichcontains BG or has a possibility of containing BG is used as the“LAM-containing sample” in the BG-measuring method of the presentinvention.

The mycosis to be detected is not particularly limited, so long as it isa disease which is classified into the category of mycosis. Examplesinclude deep fungal infection and the like.

Explanation for the “sample derived from the living body” is the same asin the Et-related disease detection method of the present invention inthe above-described <7>. It is also the same as the above-described <7>that when blood is used as the “sample derived from the living body”, itis preferable to remove or inactivate Limulus reaction-interferingfactors in blood (serine protease, a serine protease inhibitor, etc.) inadvance by a conventionally known method (e.g., the method described inJP-A-58-85162, etc.).

Mycosis can be detected in such a sample by using the BG-measuringmethod of the present invention.

Explanation for the “detection” and the like are also the same as in theabove-described <7>.

<12> BG-Measuring Kit of the Present Invention

The BG-measuring kit of the present invention is a kit for measuring BG,which comprises a Limulus reagent and one or more substance(s) selectedfrom the following group as components:

a surfactant, an anti-tuberculosis antibody, an anti-LAM antibody, astrong alkaline substance, polymyxin B, colistin, concanavalin A,histidine and histamine.

Explanations for the “Limulus reagent” and the above-described varioussubstances are the same as in the above-described <1> and <5>. However,it is preferable that the Limulus reagent to be used herein is aBG-specific Limulus reagent.

The BG-measuring kit of the present invention may further comprise othercomponents, so long as it comprises at least the “Limulus reagent” as acomponent. Explanation for such components and the like are the same asin the Et-measuring kit of the present invention of the above-described<8>.

BG using the BG-measuring kit of the present invention can be measuredin accordance with the BG-measuring method of the present invention ofthe above-described <10>.

<13> Mycosis Detection Kit of the Present Invention

The mycosis detection kit of the present invention is a kit fordetecting mycosis, which comprises the BG-measuring kit of the presentinvention. Explanation for the BG-measuring kit of the present inventionshould be referred to the above-described <12>.

Explanation for the mycosis to be detected by the mycosis detection kitof the present invention, explanation of the “sample derived from theliving body” to be used and the like are the same as in the mycosisdetection method of the present invention of the above-described <11>.That is, the mycosis to be detected includes a deep fungal infection,and it is preferable that the “sample derived from the living body” tobe used is blood. In addition, it is also the same as in theabove-described <11> that when blood is used as the “sample derived fromthe living body”, it is preferable to remove or inactivate Limulusreaction-interfering factors in blood (serine protease, a serineprotease inhibitor, etc.) in advance.

Mycosis using the mycosis detection kit of the present invention can bedetected in accordance with the mycosis detection method of the presentinvention of the above-described <11>.

<14> Agent for Binding of the Present Invention

The invention further provides an agent for binding of LAM, whichcomprises one or more substance(s) selected from the following group asan active ingredient (hereinafter referred to as “agent for binding ofthe present invention”):

an anti-tuberculosis antibody, an anti-LAM antibody, (1→3)-β-glucan, acarboxymethylated (1→3)-β-glucan, a factor G activation inhibitor,polymyxin B, colistin, concanavalin A, histidine and histamine.

Explanation for the above-described various substances is the same as inthe above-described <5>.

In addition, the agent for binding of the present invention may containanother substance, so long as it contains at least one of theabove-described substrate group as an active ingredient. The “anothersubstance” as used herein is not particularly limited, so long as itdoes not substantially spoil the LAM-binding action of the substance asan active ingredient of the agent for binding of the present invention.Examples of such an “another substance” include those which do notsubstantially spoil the LAM-binding action of the active ingredientsubstance of the agent for binding of the present invention, amongfillers, buffers, stabilizers, preservatives and the like which are usedin the preparation of general medicines or reagents.

In this connection, the term “agent for binding of LAM” as used hereinmeans an agent which is used for the purpose of binding the activeingredient substance in the preparation to LAM.

In addition, since the active ingredient substance binds to LAM, theagent for binding of the present invention can, for example, be used inthe detection and measurement of LAM, removal of LAM and the like.

That is, LAM can be detected or measured by allowing the agent forbinding of the present invention to contact with LAM (allowing amolecule of the active ingredient substance of the agent for binding ofthe present invention to contact with a LAM molecule), and thendetecting the active ingredient substance bonded to LAM, directly orafter its elution. In this case, the active ingredient substance in theagent for binding of the present invention may be labeled with asubstance which can be detected as a certain specific signal (e.g.,enzyme, radioisotope, fluorescence dye, chemiluminescence substance,hapten, metal particle, specific coupling pair, etc.). The labelingmethod and its detection and measuring methods are not particularlylimited too, and conventionally known techniques can be used.

In addition, LAM in a solution can be removed by allowing the agent forbinding of the present invention to adhere to an insoluble carrier(allowing a molecule of the active ingredient substance in the agent forbinding of the present invention to form a solid phase on an insolublecarrier), binding LAM to the active ingredient substance molecule madeinto a solid phase by allowing this to contact with a solutioncontaining LAM, and then separating the carrier to which the activeingredient substance has been adhered, from the solution. The carrier towhich the active ingredient substance in the agent for binding of thepresent invention is adhered and the adhering method are notparticularly limited, and conventionally known techniques can be used.

Accordingly, the present invention also includes general ideas such as amethod for detecting or measuring LAM, which comprises using one or moresubstance(s) selected from the following group, a kit for measuring LAM,which comprises one or more substance(s) selected from the followinggroup as a component(s), a method for removing LAM, which comprisesusing a carrier to which one or more substance(s) selected from thefollowing group are adhered, and an agent for removing LAM (adsorptionremoval agent), which comprises a carrier to which one or moresubstance(s) selected from the following group are adhered:

an anti-tuberculosis antibody, an anti-LAM antibody, (1→3)-β-glucan, acarboxymethylated (1→3)-β-glucan, a factor G activation inhibitor,polymyxin B, colistin, concanavalin A, histidine and histamine.

<15> Examples of the Illustrative Embodiments of the Present Invention

For example, all of specific embodiments shown below are included in thepresent invention. In this connection, the following embodiments aresimple illustrations, and technical scope of the present invention isnot restricted thereby.

-   (1) A method for measuring Et activity (concentration) in a    LAM-containing sample without influence of the coexisting LAM,    wherein the sample is allowed in advance to contact with at least    one substance selected from a surfactant, pachyman, an    antituberculous dead cell antibody and an anti-LAM antibody, and    then measured using an Et-specific Limulus reagent.-   (2) A method for measuring LAM activity (concentration) in a    LAM-containing sample without influence of the coexisting Et,    wherein the sample is heated in advance, and then measured using an    Et-specific Limulus reagent.-   (3) A method for detecting endotoxemia or Gram-negative bacterial    infection by measuring Et concentration in blood by the method of    the above-described (1).-   (4) A method for detecting tubercle bacillus infection by measuring    LAM concentration in blood by the method of the above-described (2).-   (5) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with a    surfactant and a factor G activation inhibitor, and then measured    using a Limulus reagent which reacts with both of Et and BG.-   (6) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with    pachyman and a factor G activation inhibitor, and then measured    using a Limulus reagent which reacts with both of Et and BG.-   (7) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with an    antituberculous dead cell antibody and a factor G activation    inhibitor, and then measured using a Limulus reagent which reacts    with both of Et and BG.-   (8) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with an    anti-LAM antibody and a factor G activation inhibitor, and then    measured using a Limulus reagent which reacts with both of Et and    BG.-   (9) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with a    surfactant and curdlan (and/or carboxymethylcurdlan), and then    measured using a Limulus reagent which reacts with both of Et and    BG.-   (10) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with    pachyman and curdlan (and/or carboxymethylcurdlan), and then    measured using a Limulus reagent which reacts with both of Et and    BG.-   (11) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with an    antituberculous dead cell antibody and curdlan (and/or    carboxymethylcurdlan), and then measured using a Limulus reagent    which reacts with both of Et and BG.-   (12) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is allowed in advance to contact with an    anti-LAM antibody and curdlan (and/or carboxymethylcurdlan), and    then measured using a Limulus reagent which reacts with both of Et    and BG.-   (13) A method for measuring Et activity (concentration) in a    LAM-containing sample without influences of the coexisting LAM and    BG, wherein the sample is heated in advance in the presence of a    factor G activation inhibitor, and then measured using a Limulus    reagent which reacts with both of Et and BG.-   (14) A method for measuring LAM activity (concentration) in a    LAM-containing sample without influences of the coexisting Et and    BG, wherein the sample is heated in advance in the presence of    curdlan and/or carboxymethylcurdlan, and then measured using a    Limulus reagent which reacts with both of Et and BG.-   (15) A method for detecting tuberculous infection by measuring LAM    activity (concentration) in blood, using plasma or serum as the    “LAM-containing sample” in the above-described (13) and (14).-   (16) A method for measuring Et activity (concentration) in blood    without influences of the coexisting LAM and BG and thereby    detecting endotoxemia or Gram-negative bacterial infection, wherein    plasma or serum is diluted in advance with a surfactant and aqueous    solutions containing “at least one substance selected from pachyman,    an antituberculous dead cell antibody and an anti-LAM antibody” and    “at least one substance selected from an factor G activation    inhibitor, curdlan and carboxymethylcurdlan”, heated and then    measured using a Limulus reagent which reacts with both of Et and    BG.-   (17) A method for measuring Et activity (concentration) in blood    without influences of the coexisting LAM and BG and thereby    detecting endotoxemia or Gram-negative bacterial infection, wherein    plasma or serum is diluted in advance with a surfactant and an    aqueous solution containing “at least one substance selected from    pachyman, an antituberculous dead cell antibody and an anti-LAM    antibody”, heated and then measured using an Et-specific Limulus    reagent.-   (18) A method for detecting a deep fungal infection, wherein BG    activity (concentration) in blood is measured without influences of    the coexisting LAM and Et, by heating plasma or serum in advance in    the presence of polymyxin B and then measuring it using a Limulus    reagent which reacts with both of Et and BG.-   (19) A method for detecting or measuring LAM, by allowing a molecule    of the active ingredient substance (labeled) of the agent for    binding of the present invention to contact with a LAM molecule, and    then detecting the substance (the label) bonded to LAM directly or    after elution.-   (20) A method for removing LAM in a solution, by making a solid    phase of a molecule of the active ingredient substance of the agent    for binding of the present invention onto an insoluble carrier,    allowing this to contact with a solution containing LAM, and then    removing the carrier by separating from the solution.

Examples of the present invention are specifically described below.However, the technical scope of the present invention is not limitedthereto.

In this connection, the LAM used in the examples is an LAM (availablefrom Nacalai Tesque) isolated and purified to a high purity from deadcells of a strain of a human type tubercle bacillus (Aoyama B strain;Mycobacterium tuberculosis Aoyama-B) by organic solvent extraction andcolumn chromatography.

EXAMPLE 1

This LAM was dissolved in distilled water (Et- and BG-free) to aconcentration of 200 ng/ml to prepare a LAM-containing sample. Thissample was subjected to each of the following various treatments, andthen 25 μl of the sample was mixed with 100 μl of an Et-specific Limulusreagent (trade name: Endospec ES Test MK, available from SEIKAGAKUCORPORATION) in an Et- and BG-free microtiter plate (Toxipet Plate 96F,available from SEIKAGAKU CORPORATION), and measured at 37° C. for 30minutes in a microplate reader (Wellreader SK 603, available fromSEIKAGAKU CORPORATION).

Thereafter, the degree of the Limulus reaction was calculated from theratio of change (mAbs/min) of absorbance [A405 nm-492 nm (controlwavelength)] per minute and compared with that of an untreated sample(control).

In this connection, the Limulus reagent used herein had been treated insuch a manner that factor C alone can be activated by removing thereactivity of factor G (treated such that it reacts only with Et).Accordingly, when the Limulus reaction was detected, it means that thefactor C was activated (that the added sample has the ability toactivate factor C).

Treatments:

Polymyxin B Treatment:

The same amount of polymyxin B sulfate (Sigma) was added to aLAM-containing sample to a final concentration of 1 mg/ml and mixed.

Acid Heating Treatment:

The same amount of HCl aqueous solution was added to a LAM-containingsample to a final concentration of 0.2 M, mixed and incubated at 37° C.for 60 minutes.

Alkali Heating Treatment:

The same amount of KOH aqueous solution was added to a LAM-containingsample to a final concentration of 0.2 M, mixed and incubated at 37° C.for 60 minutes.

Boiling Treatment:

A LAM-containing sample was boiled for 60 minutes.

Surfactant Treatment:

The same amount of each of various nonionic surfactants (Brij 56,Triton-N-101, Triton X-405, Triton X-114 and Tergitol NP-9) was added toa LAM-containing sample to a final concentration of from 0.005 to 0.5%and mixed.

BG Treatment:

The same amount of pachyman was added to a LAM-containing sample to afinal concentration of 50 pg/ml and mixed.

Concanavalin A Treatment:

To 0.25 g in wet weight of Concanavalin A (Con A) Sepharose 4B(available from Amersham Bioscience), 1 ml of an LAM aqueous solutionwas added, mixed and then centrifuged at 3,000 rpm for 10 minutes, andthe thus obtained supernatant was used.

Reactivity with an Anti-Tuberculosis Antibody:

Reactivity of LAM with an anti-tuberculosis antibody (an anti-human typetubercle bacillus dead cell monoclonal antibody) was examined.

Anti-Factor C Antibody Treatment:

A “solution prepared by adding 140 μl of 20 times-diluent of a mouseanti-factor C monoclonal antibody (2C12; ascites) to 1 vial of anEt-specific Limulus reagent (trade name: Endospec ES Test MK, availablefrom SEIKAGAKU CORPORATION)” was added to a LAM-containing sample and aLimulus reaction was carried out.

The results are shown in Table 1. In this connection, the “+” in Table 1shows that the reaction was detected, and the illustrative numericalvalues in Table 1 mean relative values when the reactivity in theuntreated sample (control) was defined as 100%.

TABLE 1 Untreated sample (control) + Polymyxin B treatment  7.5% Acidheating treatment 72.0% Alkali heating treatment   0% Boiling treatment44.4% Surfactant treatment   0% BG treatment  1.6% Concanavalin Atreatment  5.7% Reactivity with anti-tuberculosis antibody + Anti-factorC antibody treatment   0%

Based on this result, it was found that LAM has the reactivity with aLimulus reagent (has the ability to activate the factor C). Accordingly,it was shown that measurement of LAM and detection of acid-fast bacteriakeeping LAM as a cell wall component are possible by using the Limulusreagent.

Also, it was found that the Limulus reagent reactivity owned by LAM canbe removed or reduced by using polymyxin B, a strong alkaline substance,various nonionic surfactant, BG, concanavalin A and the like.

Also, since it was confirmed that LAM reacts with an anti-tuberculosisantibody, it was suggested that the reactivity of LAM with a Limulusreagent can also be removed or reduced by allowing a LAM-containingsample with the anti-tuberculosis antibody in advance.

In addition, since it was shown that LAM binds to an anti-tuberculosisantibody, concanavalin A and the like, it was indicated that removal ofLAM and the like can also be carried out using them.

Accordingly, it was shown that Et and BG can be measured out withoutinfluence of LAM, by treating a LAM-containing sample with suchsubstances. In addition, it was shown that Et-related diseases andmycosis can also be detected by this without influence of LAM. Further,it was shown also that an anti-tuberculosis antibody, concanavalin A andthe like can be used as agents for binding of LAM and can be used forthe removal of LAM and the like.

EXAMPLE 2

(1) Production Example of the LAM-Measuring Kit of the Present Invention(Acid-Fast Bacteria Detection Kit of the Present Invention)

A kit containing the following reagents was prepared.

A. Et-Specific Chromogenic Limulus Reagent

A freeze-dried product comprising a lysate prepared from blood cell(amoebocyte) of a horseshoe crab (Tachypleus tridentatus) in accordancewith the method described in Reference Example 3 of WO 90/02951, fromwhich the reactivity of factor G was removed, a coloring syntheticsubstrate (Boc-Leu-Gly-Arg-pNA) and the like.

B. Distilled Water (Et-Free)

This is used in the blank test, for the dissolution and dilution ofpositive control, dilution of test samples and the like.

C. Buffer

0.2 mol/l Tris-HCl buffer (pH 8.0). This is used for the dissolution ofthe Limulus reagent and in the reaction.

D. Positive Control

A freeze-dried product containing LAM.

LAM (an acid-fast bacterium) in a test sample can be measured anddetected without influence of Et, by heating the test sample in advanceby the method described in the above-described “<1> LAM-measuring methodof the present invention” and then carrying out the Limulus reactionusing this kit.

(2) Production Example of the Et-Measuring Kit of the Present Invention(Et-Related Disease Detection Kit of the Present Invention)

A kit containing the following reagents was prepared.

A. Et-Specific Chromogenic Limulus Reagent

Endospec ES Test MK (trade name; available from SEIKAGAKU CORPORATION)is used.

B. Distilled Water (Et-Free)

This is used in the blank test, for the dissolution and dilution ofpositive control, dilution of test samples and the like.

C. Buffer

0.2 mol/l Tris-HCl buffer (pH 8.0). This is used for the dissolution ofthe Limulus reagent and in the reaction.

D. Positive Control

A freeze-dried product containing Et derived from an Escherichia coli0111:B4.

E. Blood Pre-Treating Solution

-   First solution: 0.2 mol/l KOH, 0.2% polybrene-   Second solution: 0.2% Triton X-100, 0.14% ethyleneimine polymer,    0.02 mol/l CaCl₂, 0.06 mol/l Bicine

This pre-treating solution is used when plasma or serum is used as atest sample, in order to inactivate Limulus reaction-interfering factorscontained therein.

The first solution is mixed with the second solution immediately beforeuse.

F. Surfactant

An aqueous solution containing 0.01% Tergitol NP-9.

The surfactant is used for removing the Limulus reagent reactivity ofLAM. That is, Et (Et-related disease) can be measured and detectedwithout influence of LAM, by treating a test sample (treated with theblood pre-treating solution in the case of a blood sample) in advancewith this surfactant and then carrying out the Limulus reaction.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese patent application No. 2003-425472filed on Dec. 22, 2003, the entire contents of which are incorporatedhereinto by reference. All references cited herein are incorporated intheir entirety.

Industrial Applicability

The LAM-measuring method of the present invention, the acid-fastbacterium detection method of the present invention, the LAM-measuringkit of the present invention and the acid-fast bacterium detection kitof the present invention can be applied to the measurement and detectionof LAM and acid-fast bacteria.

In addition, the reactivity-removing method of the present invention canbe applied to the removal of influence of LAM in the Limulus reaction.

Also, the Et-measuring method of the present invention, the Et-relateddisease detection method of the present invention, the Et-measuring kitof the present invention and the Et-related disease detection kit of thepresent invention can be applied to the measurement and detection of Etand Et-related diseases.

Also, the BG-measuring method of the present invention, the mycosisdetection method of the present invention, the BG-measuring kit of thepresent invention and the mycosis detection kit of the present inventioncan be applied to the measurement and detection of BG and mycosis.

In addition, the agent for binding of the present invention can be usedin the detection and measurement of LAM, the kit to be used therein, theremoval method of LAM, the LAM-removing agent (an adsorption removalagent) and the like.

The invention claimed is:
 1. A method to detect lipoarabinomannan (LAM) in a LAM-containing sample without influence of the coexisting endotoxin (Et), wherein the sample is heated in advance, and then detected using an Et-specific Limulus reagent comprising: heating the lipoarabinomannan-containing sample in a temperature range of 37° C. to 121° C.; keeping the heated state for 5 to 60 minutes before contact with an endotoxin-specific Limulus reagent; contacting the heated sample containing lipoarabinomannan with an endotoxin-specific Limulus reagent; wherein the LAM in said sample activates factor C in the Limulus reagent, thereby inducing a Limulus reaction; and detecting the limulus reaction to detect the LAM in the sample. 